Table 3.

Recently developed in vivo biofilm models

Description	Application	Microorganism(s)	Biofilm analysis	Outcome(s)
Recent in vivo biofilm models for tissue-related infection
Endophthalmitis (Sadaka et al., 2014)
The anterior chamber of the right eye is inoculated with S. aureus using a 35-G needle on a NanoFil syringe just anterior to the limbus without touching the iris. The left eye is used as an untreated control. Animals: ♀C57BL/6J mice	Investigate the impact of CodY deletion on S. aureus virulence in a murine anterior chamber infection model	S. aureus (MS7/SA564/CDM7) Initial cell density: 1 μL of OD600nm = 0.4–0.8	•Bacterial growth assessed after 24 h by extraction and homogenization of the entire eye followed by quantitative plating •Light microscopy of histological samples	•Developed a model of anterior chamber infection, characteristic of endophthalmitis •Revealed a link between branched-chain amino acid responsive transcription regulator CodY and endophthalmitis repression •Addition of branched-chain amino acids to postoperative eyedrops could reduce progression of endophthalmitis
Keratitis (Saraswathi and Beuerman, 2015)
Four superficial abrasions of 1–2 mm are introduced into the corneal epithelium to which an aliquot of P. aeruginosa is then delivered Animals: 7–8-week-old C57BL/6 mice	Follow the progression of corneal biofilms from planktonic to microcolony formation in a corneal injury model	P. aeruginosa (ATCC 9027) Initial cell density: 5 μL of 1 × 108 CFU/mL	•Slit lamp •Light microscopy •CLSM •SEM •TEM	•Developed a model for studying biofilm infections of the corneal surface induced by P. aeruginosa •Suggests that mature biofilms are a common component of keratitis •Biofilm formation explains occasional resistance toward therapeutic treatments
Keratitis (Ponce-Angulo et al., 2020)
A micropocket incision in the limboscleral border of the cornea is made to which a bacterial inoculate is injected Animals: 6–8-week-old ♀BALB/c mice	Characterize and analyze biofilm formation in mixed keratitis induced by coinfection	S. aureus (IOM2617228) F. falciforme (IOM325286) Initial cell density (CFU/mL): S. aureus 3 μL of 1 × 105 F. falciforme 8 μL of 1 × 105	•Histopathological analysis via optical and fluorescence microscopy using Gomori-Grocott and periodic acid-Schiff staining •TEM	•Developed a model for studying mixed biofilm infections of the corneal surface
Vaginosis (Nash et al., 2016)
Intravaginal infections are induced through bacterial inoculation Animals: 8–10-week-old ♀C57BL/6 mice, age matched C3H/HeN mice (KK.Cg-AY/J), 9–11-week-old KK.Cg-AY/J mice	Test whether C. glabrata like C. albicans results in inflammatory immunopathogenic vaginitis in a diabetic mouse model	C. glabrata (BG2) C. albicans (DAY185) Initial cell density: C. glabrata 2 × 106−1 × 107 CFU C. albicans 5 × 104−5 × 106 CFU	•Colonization was assessed by quantitative culture of vaginal swabs •Confocal microscopy of excised and stained vaginal tissue	•Developed a model for studying vaginal infections caused by C. glabrata •Determined that C. glabrata does not elicit the same immunopathology as C. albicans during vaginal colonization •C. glabrata does not enhance C. albicans pathogenesis
Vaginosis (Hymes et al., 2013)
Intravaginal infections are induced through inoculation with G. vaginalis along with DNase or gelatin as control Animals: 8-week-old ♀C57BL/6J mice	Demonstrate that eDNA secreted from G. vaginalis biofilms is critical for the structural integrity of newly forming and established biofilms	G. vaginalis (ARG37) Initial cell density: 5 × 106 CFU	•Colonization was assessed by quantitative culture of vaginal swabs	•Developed a model for studying vaginal infections caused by G. vaginalis •Illustrated that intravaginal treatment with DNase inhibits de novo biofilm formation while also liberating G. vaginalis from existing biofilms without killing, a process that decreases biofilm density and could potentially enhance the effect of antimicrobials
Meningitis (Grumbein et al., 2016; Zhang et al., 2018)
Injection of a bacterial inoculate through the intracranial subarachnoidal route of infection. Injection site is located 3.5 mm rostral from the bregma Animals: 6-week-old ♀ BALB/c mice	Investigate the roles of S. suis biofilms in meningitis	S. suis (P1/7) Initial cell density: 3 × 107 CFU	•Colonization was assessed by quantitative culture •Light microscopy of histopathological samples	•Developed a model to study the mechanism of S. suis meningitis and the pathogenesis of other meningitis causing bacteria as well as the efficacy of new drugs against bacterial meningitis •Showed that disruption of the blood-brain or blood-cerebrospinal fluid barrier by S. suis are important steps in the development of meningitis
Chronic abscess infections (Pletzer et al., 2017)
Bacteria are injected onto the right side of the dorsum underneath the thin skeletal muscle Animals: 7-week-old ♀ (25 ± 2 g) & ♂ (35 ± 5 g) CD-1 mice, 15-week-old ♀ (35 ± 5 g) CD-1 mice, 7-week-old ♀ & ♂ (17± 2 g) C57BL/6 mice	Test the efficiency of treatments and drugs against chronic subcutaneous infections caused by Gram-negative bacteria	(1)P. aeruginosa (LESB58, PA14) (2)A. baumanni (AB5075) (3)K. pneumaniae (KPLN49) (4)E. coli (MG1655) (5)E. cloacae (218R1) Initial cell density (CFU): (1) 50 μL of 107; 50 μL of 106; (2), (3), & (4) 50 μl of 108; (5) E. cloacae 50 μL of 107	•Progression of the infection was monitored by measuring the size of the abscess lesion •Skin abscesses were excised and homogenized to determine bacterial counts by serial dilution •Histology, qPCR, and IVIS	•Developed a murine model that can be used as a rapid and easy in vivo secondary screening assay for testing novel compounds, enabling toxicity studies, and determining their efficacy against a variety of Gram-negative bacteria •Testing of organs showed that infections did not disseminate
Recent in vivo biofilm models for device-related infection
Cochlear implant (Cevizci et al., 2015)
Bacteria are instilled into the middle ear Guinea pigs through the tympanic membrane. Small pieces of cochlear implant are then implanted under the skin in the retroauricular area after being soaked in a pneumococcal solution Animals: 500–600 g ♀ & ♂ Dunkin Hartley Guinea pigs	Assess the effect of a QS inhibitor against otitis media and biofilm formation on cochlear implants	S. pneumoniae (19F) Initial cell density: 500 μL of 1 × 105 CFU	•SEM	•Developed a model for cochlear implant infection •Illustrated a novel QS inhibitor that could prevent biofilm formation on cochlear implants
Neurological device (Glage et al., 2017)
Two holes are drilled in the cranium. The holes are inoculated with bacteria and then fitted with titanium screws Animals: 210–260 g ♀ Sprague-Dawley rats	Test whether intraoperative infection of intracranial screws would lead to biofilm formation and inflammatory tissue reaction	S. aureus (36/07, ZTL) Initial cell density: 5 μL of 1 × 107 CFU	•Bacterial growth and biofilm formation on the screws was analyzed by CLSM after staining with propidium iodide and Syto 9	•Developed a model for cranial implant infection •Allows for the study of pathophysiologic mechanisms leading to implant failure, test novel therapeutic interventions, antibacterial coatings, and novel materials
Neurological device (Snowden et al., 2012)
Catheters are colonized with bacteria and then placed into the lateral brain ventricle. After catheter placement, the burr hole is sealed with bone wax, and the incision sealed with surgical tissue glue Animals: 8–9-week-old ♂ C57BL/6 mice	Characterize the nature of the inflammatory response during biofilm growth within central nervous system catheter	S. aureus (MSSA) Initial cell density: 2 × 104 CFU/mL	•SEM of catheters after removal •Quantitative bacterial plating of catheters and surrounding homogenized tissue	•Developed a model of central nervous system catheter infection •Provides tool for screening patients at high risk of infection and testing adjunctive therapies to current antibiotic treatment regimens